Culture method for promoting proliferation and rejuvenation of subculture buds of leucaena fragrans

Abstract

The invention discloses a culture method for promoting proliferation and rejuvenation of subculture buds of leucaena fragrans, which comprises the following steps: dedifferentiation: taking the subculture buds after 22 generations, cutting into tissue blocks, inoculating into a callus induction culture medium, and inducing dedifferentiation under preset induction conditions to form calluses for 14-20 days; inducing cluster buds: cutting the callus, inoculating the cut callus into a differential culture medium, and inducing the cluster buds under preset differentiation conditions; rejuvenation culture: taking stems or buds of the cluster buds, inoculating the stems or buds into a rejuvenation culture medium, and culturing under preset culture conditions to obtain rejuvenated seedlings; and subculture: cutting stem segments of the compound strong seedlings, inoculating the stem segments into subculture, and culturing under preset culture conditions. Compared with the prior art, the method has the advantages that by means of inducing the subculture buds after 22 generations to form calluses, inducing the cluster buds, rejuvenating and subculturing, the subculture buds of the albizia julibrissin with a decline phenomenon are re-differentiated, regenerated and reproductive capacity, and the effect of prolonging the tissue culture life of the subculture buds of the albizia julibrissin is achieved.

Description

Technical Field

[0001] The invention relates to plant tissue culture, and in particular to a culture method for promoting proliferation and rejuvenation of successive buds of Albizzia serrata. Background Art

[0002] Albizia odoratissima (Lf) Benth, also known as Niujiaosen and Heige, is a tall deciduous tree of the genus Albizia in the Fabaceae family. Currently, due to urban expansion and weak awareness of protection, wild Albizia odoratissima has been severely cut down, and the natural regeneration of Albizia odoratissima is difficult to complete smoothly. In order to preserve this tree species or quickly obtain high-quality seedlings, our institute has conducted a lot of research on Albizia odoratissima in plant tissue culture, such as:

[0003] CN202010880528.8 discloses a method for rapid propagation of the high-value timber tree species Albizzia julibrissin through tissue culture. Specifically, the steps of disinfection of Albizzia julibrissin explants, primary induction, secondary proliferation culture, rooting culture, seedling hardening and transplanting, and seedling transplanting are disclosed to achieve an initial bud induction rate of more than 97% and a cluster bud induction rate of more than 98%, achieving a very good induction effect.

[0004] CN202010880492.3 discloses a method for effectively promoting the subculture proliferation culture of Albizia Julibrissin, specifically disclosing the processes of selection and treatment of Albizia Julibrissin explants, primary induction, subculture proliferation culture, etc. to achieve an initial bud induction rate of more than 98% and a cluster bud induction rate of more than 99%, achieving a good induction effect.

[0005] Plant tissue culture is an important way to preserve species genetic resources and rapidly propagate to obtain plant seedlings. However, through in-depth research on tissue culture of Albizzia julibrissin, this research team found that there is a problem with the buds showing a decline trend after multiple subcultures in tissue culture of Albizzia julibrissin. Specifically, the subculture of Albizzia julibrissin showed decline after 22 generations. The decline of subculture buds of Albizzia julibrissin was manifested by slow growth after inoculation, easy yellowing, and decreased differentiation and regeneration ability, resulting in poor tissue culture effect and low quality of tissue culture seedlings. However, the steps of re-using explants for tissue culture are cumbersome and time-consuming. Therefore, extending the lifespan of subculture buds of Albizzia julibrissin is of great significance to the development of tissue culture technology of Albizzia julibrissin. Summary of the invention

[0006] The purpose of the present invention is to provide a culture method for promoting the proliferation and rejuvenation of the succession buds of Albizzia frasii, aiming to overcome the phenomena of slow growth, yellowing, browning of callus tissue, and decreased differentiation and regeneration reproduction ability of the succession buds of Albizzia frasii after 22 generations, restore the differentiation and regeneration reproduction ability of the succession buds of Albizzia frasii which show a declining trend, and prolong the tissue culture life of the succession buds of Albizzia frasii.

[0007] To achieve the above object, the present invention provides a culture method for promoting the proliferation and rejuvenation of Albizzia julibrissin subgeneration buds, the method comprising:

[0008] S1, dedifferentiation: take the 22nd generation successive buds, cut them into tissue blocks, inoculate them into callus induction medium, and induce dedifferentiation to form callus under preset induction conditions for 14-20 days, wherein the callus induction medium consists of modified WPM medium, 0.3-0.9 mg / L methyl jasmonate, 0.8-1.6 mg / L LTDZ, 0.4-1.2 mg / L NAA, 1.0-2.0 mg / L IBA, 1.5-2.5 g / L sucrose and 4.8-7.5 g / L agar;

[0009] S2, inducing clustered buds: cutting the callus tissue, inoculating the cut callus tissue into a differentiation medium, and placing it under a preset differentiation condition to induce clustered buds;

[0010] S3, rejuvenation culture: taking the stems or buds of the clustered buds and inoculating them into a rejuvenation medium, placing them under preset culture conditions and culturing them to obtain a rejuvenation seedling, wherein the rejuvenation medium comprises WPM medium, 0.2-0.6 mg / L TDZ, 0.4-0.8 mg / L IBA, 0.3-0.8 mg / L methyl jasmonate, 0.5-1 mg / L chitin, 0.1-0.5 mg / L riboflavin sodium phosphate, 3-6 g / L sucrose and 4.8-7.5 g / L agar;

[0011] S4, subculture: Cut the stem segments of the rejuvenated seedlings and inoculate them into subculture, and culture them under the preset culture conditions.

[0012] Preferably, in the above technical solution, the improved WPM culture medium is composed of 70-76 mg / L CaCl2, 470-475 mg / L Ca(NO3)2·4H2O, 170-175 mg / L KH2PO4, 340-360 mg / L K2SO4, 360-380 mg / L MgSO4, 180-210 mg / L KNO3, 0.2-0.3 mg / L CuSO4·5H2O, 40-50 mg / LFeNaEDTA, 2-4 mg / L H3BO3, 8-11 mg / LMnSO4·H2O, 0.1-0.15 mg / L It is made of Na2MoO4·2H2O, 10-14mg / LZnSO4·7H2O, 0.5-0.8mg / L niacin, 180-220mg / L inositol, 0.4-0.6mg / L vitamin B6 and 1-2mg / L vitamin B1.

[0013] Preferably, in the above technical solution, the preset induction conditions include a temperature of 24-28°C, a light intensity of 300-600Lx, and a lighting time of 12-14h / d.

[0014] Preferably, in the above technical solution, in step S2, the specific steps of cutting the callus tissue and inoculating the cut callus tissue into a differentiation medium are:

[0015] The callus tissue is cut into 3-5 mm callus tissue blocks, and the callus tissue blocks are half-inserted into a differentiation medium, wherein the differentiation medium is composed of WPM medium, 0.8-2.4 mg / L TDZ, 0.3-0.8 mg / L methyl jasmonate, 0.4-1.6 mg / L IBA, 0.5-1 mg / L brassinolide, 0.1-0.8 mg / L La(NO3)3, 3-6 g / L sucrose and 4.8-7.5 g / L agar.

[0016] Preferably, in the above technical solution, the preparation method of the differentiation medium is:

[0017] (1) preparing a mixed solution of WPM medium, TDZ, methyl jasmonate, IBA, and La(NO3)3, and completely dissolving sucrose and agar in water by heating, then adding the mixed solution to adjust the pH value to 6.5-6.8, fixing the volume, and dispensing into tissue culture bottles, and sterilizing the medium by high-pressure steam sterilization;

[0018] (2) preparing a 0.5-1 mg / L brassinolide solution, heating the 0.5-1 mg / L brassinolide solution in a water bath to 75-80° C., and maintaining the temperature for 25-35 min to obtain a sterile 0.5-1 mg / L brassinolide solution;

[0019] (3) Mix the brassinolide solution with the unsolidified culture medium next to the flame of an alcohol lamp on a clean bench, and let it stand to solidify to obtain a differentiation culture medium.

[0020] Preferably, in the above technical solution, the preset differentiation conditions include a temperature of 24-28°C, a light intensity of 3000-4000Lx and a light intensity of 16-18h / d.

[0021] Preferably, in the above technical solution, in step S4, the stem segment is a robust stem retaining 1-2 stem nodes.

[0022] According to the above technical scheme, the principle of inoculating a robust stem with 1-2 stem nodes in subculture is that when the inoculation object was not limited to the stem in the previous experiment, it was found that the subculture seedlings grown unevenly, with a high proliferation coefficient but a thin and weak seedling body. After multiple observations and studies, it was speculated that the buds grew faster and proliferated more, resulting in a thin and weak seedling body. When the inoculation was a robust stem with 1 stem node, the proliferation coefficient of the tissue culture seedlings obtained by subculture was 2-4, the leaves were larger, and the stems were stronger.

[0023] Preferably, in the above technical solution, the subculture medium is composed of WPM medium, 0.5-mg / L methyl jasmonate, 0.8 mg / L NAA and 0.6 mg / L IBA.

[0024] Preferably, in the above technical solution, the preset culture conditions include a temperature of 24-28°C, a light intensity of 6000-8000Lx and a light intensity of 16h / d.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0026] (1) The subculture buds after 22 generations of Albizzia julibrissin were used as tissue culture research objects. Through inducing callus-inducing clustered buds-rejuvenation-subculture, the problems of inability to subculture and germplasm preservation caused by subculture aging in the tissue culture technology of Albizzia julibrissin were solved, and the life span of subculture buds of Albizzia julibrissin was extended;

[0027] (2) During the callus induction stage, by improving the WPM medium and adjusting the concentrations of methyl jasmonate, TDZ, sucrose and other substances, as well as by setting induction conditions, the dedifferentiation of tissue blocks to form callus tissue was promoted, while the oxidation of phenolic substances such as tannins in the tissue blocks of Albizzia julibrissin into quinone structures and browning and necrosis was reduced;

[0028] (3) In the stage of inducing callus differentiation into clustered buds, a differentiation medium is prepared by separation and sterilization, and TDZ, IBA, brassinolide and La(NO3)3 are set in the differentiation medium to promote the proliferation and differentiation of callus into clustered buds;

[0029] (4) During the rejuvenation culture stage, by adding methyl jasmonate to the culture medium to reduce the browning of the inoculated tissue, increasing the concentrations of chitin and riboflavin sodium phosphate and reducing TDZ and IBA, the formation of callus tissue at the inoculation site and the proliferation coefficient were reduced, so that the subculture seedlings were thicker and the length between stems was appropriate, avoiding the excessive growth of the tissue culture seedlings, and prolonging the tissue culture life of the subculture buds of Albizzia julibrissin. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The realization of the purpose, functional features and advantages of the present invention will be further explained in conjunction with embodiments and with reference to the accompanying drawings.

[0031] Figure 1 This is a picture of the overall growth of the tissue culture seedlings of the same batch of 22 subsequent generations inoculated by our research team after germination;

[0032] Figure 2 Figure A in the middle shows the situation of slow growth and yellowing of tissue culture seedlings after germination in the 22nd generation;

[0033] Figure 2 Figure B in the middle shows the low proliferation coefficient of the tissue culture seedlings after germination in the 22nd generation;

[0034] Figure 3 Figure A in the figure is a figure showing that the callus induction effect of comparative example 1 is poor;

[0035] Figure 3 Figure B is a diagram showing the callus induction effect obtained by using the S1 dedifferentiation method in Example 3 of the present invention;

[0036] Figure 4 This is a diagram of obtaining tissue culture seedlings using the S2 bud induction method of the present invention;

[0037] Figure 5 is a diagram of obtaining tissue culture seedlings using the S3 rejuvenation culture method of the present invention;

[0038] Figure 6 It is a diagram of obtaining tissue culture seedlings using the S4 subculture method of the present invention. DETAILED DESCRIPTION

[0039] The decline of the successive buds of Albizzia odorifera can be seen from Figure 1 and Figure 2 see, Figure 1 The uniformity of the growth of the tissue culture seedlings obtained by inoculating the 22nd generation of successive buds is low. Some buds have lost their ability to differentiate and regenerate, resulting in the inability to cultivate new plants through some organs. Some buds have reduced their ability to differentiate and regenerate, resulting in a low proliferation coefficient (such as Figure 2 Some of the buds that regenerate from tissue culture grow slowly and turn yellow (e.g. Figure 2 (Figure A in the figure), only some buds can obtain normal tissue culture seedlings, resulting in a low input-output ratio of tissue culture, poor tissue culture effect, and low uniformity rate of tissue culture seedlings.

[0040] Example 1

[0041] A culture method for promoting the proliferation and rejuvenation of Albizzia serrata subgeneration buds, and screening a culture medium for inducing dedifferentiation of Albizzia serrata subgeneration buds.

[0042] Screening test 1:

[0043] The 22nd generation of non-chlorotic subculture buds were taken, cut longitudinally along the stem, and the tissue blocks were inoculated into callus induction medium 1 and callus induction medium 2, respectively, and placed at a temperature of 24°C, a light intensity of 300Lx, and a light intensity of 14h / d to induce dedifferentiation of subculture bud tissue for 14 days. Callus induction medium 1 was made of WPM medium + 1.6mg / L 6-BA + 0.8mg / L α-naphthaleneacetic acid (NAA) + 3g / L sucrose + 4.8g / L agar, and callus induction medium 2 was made of improved WPM medium + 1.6mg / L 6-BA + 0.8mg / L α-naphthaleneacetic acid + 3g / L sucrose + 4.8g / L agar.

[0044] The components of WPM medium and modified WPM medium are as follows:

[0045]

[0046] During the induction culture, the induction rate of callus induction medium 1 was 38%, and the induction rate of callus induction medium 2 was 51%, indicating that the improved WPM medium can effectively promote the induction of Albizia julibrissin tissue blocks. The principle of the improved WPM medium to promote induction may be that the content of K2SO4 in the macroelements is reduced, the content of MgSO4 is increased, KNO3 is used to replace NH4NO3, nitrate nitrogen is converted into ammonium nitrogen, and the tissue blocks absorb NO3 - The ions change the potential gradient, promoting the tissue block to Mg 2+ The absorption of Mg increases, which improves the dedifferentiation of Albizia Julibrissin tissue blocks to form callus tissue; secondly, it increases zinc and iron elements, participates in activating, catalyzing, and regulating enzyme activity, and promotes the dedifferentiation of tissue blocks into callus tissue.

[0047] Screening test 2:

[0048] The 22nd generation of non-chlorotic subculture buds were taken, cut longitudinally along the stem, cut into tissue blocks, and each tissue block was inoculated into callus induction medium, and placed under the conditions of 24°C, 300Lx light intensity, and 14h / d light to induce subculture bud tissue dedifferentiation for 14 days. Among them, the modified WPM medium + 1.5g / L sucrose + 4.8g / L agar was used as the basis, and TDZ (0, 0.4, 0.8, 1.6mg / L), methyl jasmonate concentration (0, 0.1, 0.5, 0.9mg / L), 6-BA concentration (0, 1, 1.5, 2.0mg / L), NAA concentration (0, 0.4, 0.8, 1.2mg / L), IBA concentration (0, 1, 1.5, 2.0mg / L) were used as the factor design. 16 The orthogonal test is shown in Table 1. Callus induction rate (%) = number of inoculations producing callus / total number of inoculations × 100%; browning rate (%) = number of inoculations that browned / total number of inoculations × 100%.

[0049] Table 1 Browning rate and induction rate of callus induction medium with different components

[0050]

[0051]

[0052] As can be seen from Table 1, methyl jasmonate can reduce the browning of Albizzia julibrissin tissue blocks, and the combination of methyl jasmonate and IBA has less browning of Albizzia julibrissin tissue blocks than the combination of methyl jasmonate and 6-BA. The principle of methyl jasmonate and IBA reducing browning is that IBA inhibits the synthesis of phenolic substances and methyl jasmonate inhibits the activity of polyphenol oxidase to reduce the browning of Albizzia julibrissin tissue blocks. The callus induction medium with the best induction effect is composed of modified WPM medium + 0.1mg / L methyl jasmonate + 1.6mg / LTDZ + 0.4mg / LNA A + 1.5mg / LIBA, with an induction rate of 85%; followed by modified WPM medium + 0.9mg / L methyl jasmonate + 0.8mg / LTDZ + 1.2mg / LNAA + 1mg / LIBA, with an induction rate of 76%.

[0053] Example 2

[0054] A culture method for promoting the proliferation and rejuvenation of Albizzia scabra subculture buds and screening a differentiation culture medium.

[0055] Take the well-growing callus from the whole stem segment and cut it into smaller callus blocks, inoculate it into the induction differentiation medium to induce differentiation and budding to obtain clustered buds. Each bottle is inoculated with 5-7 callus blocks, among which WPM medium + 3g / L sucrose + 4.8g / L agar is used as the basis. The callus blocks are placed at a temperature of 24°C, a light intensity of 4000Lx, and a light intensity of 16h / d to induce differentiation and budding of callus blocks for 35 days. TDZ (0.4, 0.8, Table 2 shows the orthogonal experiment of L16 with 5 factors and 3 levels as the factor design, including 1.6, 2.4 mg / L), methyl jasmonate concentration (0.1, 0.3, 0.5, 0.8 mg / L), IBA concentration (0.4, 0.8, 1.2, 1.6 mg / L), brassinolide concentration (0.1, 0.5, 0.8, 1.2 mg / L), and La(NO3)3 concentration (0.1, 0.5, 0.8, 1.2 mg / L).

[0056] Browning rate (%) = number of browned inoculations / total number of inoculations × 100%.

[0057] Table 2 Proliferation coefficient and induction rate of differentiation medium with different components

[0058]

[0059] It can be seen from Table 2 above that when the concentrations of TDZ and IBA are the same or the concentration of IBA is greater than the concentration of TDZ, it has a greater effect on the proliferation coefficient. When the brassinolide in the culture medium is greater than 1.0 mg / L, the proliferation coefficient changes little, and when the La(NO3)3 concentration is greater than 0.8 mg / L, the proliferation is inhibited. Therefore, the differentiation medium consists of WPM medium + 0.4-2.4 mg / LTDZ + 0.3-0.8 mg / L methyl jasmonate + 0.4-1.6 mg / LIBA + 0.1-1 mg / L brassinolide + 0.1-0.8 mg / L La(NO3)3 + 3 g / L sucrose + 4.8 g / L agar.

[0060] Example 3

[0061] A cultivation method for promoting proliferation and rejuvenation of Albizzia julibrissin subgeneration buds, the method comprising:

[0062] S1, dedifferentiation: take the 22nd generation of non-chlorotic subgeneration buds, cut them longitudinally along the stem, cut them into tissue blocks, inoculate each tissue block into callus induction medium, place them at a temperature of 24°C, a light intensity of 300Lx, and a light intensity of 14h / d to induce dedifferentiation of subgeneration bud tissue for 17 days, wherein the callus induction medium is a modified WPM medium + 0.5mg / L methyl jasmonate + 1.6mg / LTDZ + 0.4mg / L NAA + 1.5mg / LIBA + 1.5g / L sucrose + 4.8g / L agar;

[0063] S2, inducing clustered buds: taking well-growing callus, cutting the callus into 3-5 mm callus blocks, taking 5-7 callus blocks per bottle and half-inserting them into differentiation medium, placing them at a temperature of 24° C., a light intensity of 4000 Lx, and a light intensity of 16 h / d to induce callus blocks to differentiate and sprout for 35 days, and obtaining clustered buds, wherein the preparation method of the differentiation medium is as follows:

[0064] (1) preparing a mixed solution composed of WPM medium, 1.6 mg / L TDDZ, 0.5 mg / L methyl jasmonate, 0.8 mg / L IBA, and 0.5 mg / L La(NO3)3, and completely dissolving sucrose and agar in water by heating, then adding the mixed solution to adjust the pH value to 6.5-6.8, making up to volume, and dispensing into tissue culture bottles, and sterilizing the medium by high pressure steam sterilization;

[0065] (2) preparing a 0.8 mg / L brassinolide solution, heating the 0.8 mg / L brassinolide solution in a water bath to 75-80° C., and maintaining the temperature for 25 min to obtain a sterile 0.8 mg / L brassinolide solution;

[0066] (3) mixing the brassinolide solution with an unsolidified culture medium next to the flame of an alcohol lamp on a clean bench, and allowing to solidify to obtain a differentiation culture medium;

[0067] S3, rejuvenation culture: take the clustered bud ramet, retain one stem node and cut the inoculation port obliquely by 30-45 degrees, inoculate it into a rejuvenation medium, and place it under the conditions of temperature of 24°C, light intensity of 6000Lx, and light of 16h / d to induce rejuvenation of subculture buds for 45 days; the rejuvenation medium consists of WPM medium + 0.4mg / L TDZ + 0.6mg / L IBA + 0.5mg / L methyl jasmonate + 0.8mg / L chitin + 0.5mg / L riboflavin sodium phosphate + 3g / L sucrose + 4.8g / L agar.

[0068] S4, subculture: take the rejuvenated seedlings, cut the strong stems with one stem node, inoculate the stems into subculture, and place them in the subculture medium for 45 days under the conditions of temperature of 24°C, light intensity of 6000Lx, and light of 16h / d. The subculture medium consists of WPM medium, 0.5mg / L methyl jasmonate, 0.8mg / L NAA, and 0.5mg / L TDZ.

[0069] Example 4

[0070] A cultivation method for promoting proliferation and rejuvenation of Albizzia julibrissin subgeneration buds, the method comprising:

[0071] S1, dedifferentiation: take the 22nd generation of non-chlorotic subgeneration buds, cut them longitudinally along the stem, cut them into tissue blocks, inoculate each tissue block into callus induction medium, place them at a temperature of 26°C, a light intensity of 500Lx, and a light intensity of 12h / d to induce dedifferentiation of subgeneration bud tissue for 16 days, wherein the callus induction medium is a modified WPM medium + 0.3mg / L methyl jasmonate + 0.8mg / LTDZ + 0.8mg / L NAA + 1mg / L IBA + 1g / L sucrose + 6.0g / L agar;

[0072] S2, inducing clustered buds: taking well-growing callus, cutting the callus into 3-5 mm callus blocks, taking 5-7 callus blocks per bottle and half-inserting them into differentiation medium, placing them at a temperature of 26° C., a light intensity of 3000 Lx, and a light intensity of 18 h / d to induce callus blocks to differentiate and sprout for 40 days to obtain clustered buds, wherein the preparation method of the differentiation medium is as follows:

[0073] (1) preparing a mixed solution composed of WPM medium, 0.8 mg / L TDDZ, 0.5 mg / L methyl jasmonate, 0.4 mg / L IBA, and 0.1 mg / L La(NO3)3, and completely dissolving sucrose and agar in water by heating, then adding the mixed solution to adjust the pH value to 6.5-6.8, making up to volume, and dispensing into tissue culture bottles, and sterilizing the medium by high pressure steam sterilization;

[0074] (2) preparing a 0.5 mg / L brassinolide solution, heating the 0.5 mg / L brassinolide solution in a water bath to 75-80° C., and maintaining the temperature for 30 min to obtain a sterile 0.5 mg / L brassinolide solution;

[0075] (3) mixing the brassinolide solution with an unsolidified culture medium next to the flame of an alcohol lamp on a clean bench, and allowing to solidify to obtain a differentiation culture medium;

[0076] S3, rejuvenation culture: take the clustered bud ramet, retain one stem node and cut the inoculation port obliquely by 30-45 degrees, inoculate it into a rejuvenation medium, and place it under the conditions of a temperature of 26°C, a light intensity of 7000Lx, and a light intensity of 16h / d to induce rejuvenation of subgeneration buds for 50 days; the rejuvenation medium consists of WPM medium + 0.4mg / L TDZ + 0.6mg / L IBA + 0.5mg / L methyl jasmonate + 0.8mg / L chitin + 0.5mg / L riboflavin sodium phosphate + 3g / L sucrose + 4.8g / L agar.

[0077] S4, subculture: take the rejuvenated seedlings, cut the strong stems with one stem node, inoculate the stems into subculture, and place them in the subculture medium for 50 days under the conditions of temperature of 26°C, light intensity of 7000Lx, and light of 17h / d. The subculture medium consists of WPM medium, 0.3mg / L methyl jasmonate, 0.6mg / L NAA, and 0.3mg / L TDZ.

[0078] Example 5

[0079] A cultivation method for promoting proliferation and rejuvenation of Albizzia julibrissin subgeneration buds, the method comprising:

[0080] S1, dedifferentiation: Take the 22nd generation of non-chlorotic subgeneration buds, cut them longitudinally along the stem, cut them into tissue blocks, inoculate each tissue block into callus induction medium, place them in a temperature of 28°C, light intensity of 600Lx, and light of 12h / d to induce dedifferentiation of subgeneration bud tissue for 16 days, wherein the callus induction medium is a modified WPM medium + 0.9mg / L methyl jasmonate

[0081] +0.8mg / LTDZ+1.2mg / LNAA+1mg / LIBA+2g / L sucrose+7.5g / L agar;

[0082] S2, inducing clustered buds: taking well-growing callus, cutting the callus into 3-5 mm callus blocks, taking 5-7 callus blocks per bottle, half-inserting into differentiation medium, placing under the conditions of temperature of 28° C., light intensity of 4000 Lx, and light of 18 h / d to induce callus blocks to differentiate and sprout for 40 days, and obtaining clustered buds, wherein the preparation method of the differentiation medium is as follows:

[0083] (1) preparing a mixed solution composed of WPM medium, 2.4 mg / L TDDZ, 0.8 mg / L methyl jasmonate, 1.6 mg / L IBA, and 0.8 mg / L La(NO3)3, and completely dissolving sucrose and agar in water by heating, then adding the mixed solution to adjust the pH value to 6.5-6.8, making up to volume, and dispensing into tissue culture bottles, and sterilizing the medium by high pressure steam sterilization;

[0084] (2) preparing a 1 mg / L brassinolide solution, heating the 1 mg / L brassinolide solution in a water bath to 75-80° C., and maintaining the temperature for 35 min to obtain a sterile 1 mg / L brassinolide solution;

[0085] (3) mixing the brassinolide solution with an unsolidified culture medium next to the flame of an alcohol lamp on a clean bench, and allowing to solidify to obtain a differentiation culture medium;

[0086] S3, rejuvenation culture: take the clustered bud ramet, retain 1-3 stem nodes and cut the inoculation port obliquely by 30-45 degrees, inoculate it into the rejuvenation medium, and place it under the conditions of temperature of 28°C, light intensity of 8000Lx, and light of 18h / d to induce rejuvenation of subculture buds for 40 days; the rejuvenation medium consists of WPM medium + 0.8mg / LTDZ + 0.8mg / L IBA + 0.8mg / L methyl jasmonate + 1mg / L chitin + 0.5mg / L riboflavin sodium phosphate + 6g / L sucrose + 7.5g / L agar.

[0087] S4, subculture: take the rejuvenated seedlings, cut the strong stems with 2 stem nodes, inoculate the stems into subculture, and place them in the subculture medium for 40 days under the conditions of temperature of 28°C, light intensity of 8000Lx, and light of 18h / d. The subculture medium consists of WPM medium, 0.8mg / L methyl jasmonate, 1.2mg / L NAA, and 0.6mg / L TDZ.

[0088] Comparative Example 1

[0089] The dedifferentiation is basically the same as that in Example 3, except that the culture conditions are different.

[0090] Take the 22nd generation of non-chlorotic subculture buds, cut them longitudinally along the stem, cut them into tissue blocks, inoculate each tissue block into callus induction medium, and place them under the conditions of temperature of 24°C, light intensity of 4000Lx, and light of 18h / d to induce dedifferentiation of subculture bud tissue for 14d, wherein the callus induction medium is modified WPM medium + 0.5mg / L methyl jasmonate + 1.6mg / LTDZ + 0.4mg / L NAA + 1.5mg / LIBA + 1.5g / L sucrose + 4.8g / L agar.

[0091] Comparative Example 2

[0092] The dedifferentiation is basically the same as that in Example 3, except that the culture conditions are different.

[0093] Take the 22nd generation of non-chlorotic subculture buds, cut them longitudinally along the stem, cut them into tissue blocks, inoculate each tissue block into callus induction medium, and place them under the conditions of temperature of 24°C, light intensity of 4000Lx, and light of 18h / d to induce dedifferentiation of subculture bud tissue for 14d, wherein the callus induction medium is modified WPM medium + 0.5mg / L methyl jasmonate + 1.6mg / LTDZ + 0.4mg / L NAA + 1.5mg / LIBA + 4.5g / L sucrose + 4.8g / L agar.

[0094] The dedifferentiation treatment of Example 3 and Comparative Examples 1-2 was performed, and the browning and callus induction conditions were observed after 14 days of culture. The results are shown in Table 3 and Figure 3 (Figure A) as shown.

[0095] Table 3 Browning rate and induction rate of dedifferentiation of Example 3 and Comparative Examples 1-2

[0096] Example 3 Comparative Example 1 Comparative Example 2 Browning rate (%) 5 6 11 Induction rate (%) 85 76 73

[0097] It can be seen from Table 3 above that, compared with Example 3, increasing the light intensity and light exposure time in Comparative Example 1 will reduce the induction rate. Figure 3 As shown, Figure A is a figure of comparative example 1 inducing Albizzia julibrissin tissue blocks to obtain better callus tissue, and Figure B is a figure of Example 3 inducing Albizzia julibrissin tissue blocks to obtain better callus tissue. Comparison of the two shows that the callus tissue induced in each tissue block in comparative example 1 is smaller, which shows that long-term exposure of Albizzia julibrissin tissue blocks to high-intensity light during the induction of dedifferentiation will lead to a lower induction rate and smaller callus tissue blocks; compared with comparative example 1, increasing the sucrose concentration in comparative example 2 will lead to the number of browning Albizzia julibrissin tissue blocks and increase the browning rate.

[0098] Comparative Example 3

[0099] The induction of clustered shoots is basically the same as that in Example 3, except that the culture conditions are different.

[0100] Take well-growing callus from the whole stem segment and cut it into smaller callus blocks, inoculate the callus blocks into an induction differentiation medium at a rate of 5-7 blocks per bottle, place them under the conditions of a temperature of 24° C., a light intensity of 1000 Lx, and a light intensity of 14 h / d to induce the callus blocks to differentiate and sprout for 20-30 days to obtain clustered buds, wherein the differentiation medium consists of WPM medium + 1.6 mg / LTDZ + 0.5 mg / L methyl jasmonate + 0.8 mg / LIBA + 0.8 mg / L brassinolide + 0.5 mg / LLa(NO3)3 + 3 g / L sucrose + 4.8 g / L agar.

[0101] Comparative Example 4

[0102] The method of inducing clustered shoots is basically the same as that of Example 3, except that the method for preparing the differentiation medium is different. The method for preparing the differentiation medium is as follows:

[0103] A mixed solution consisting of WPM culture medium, 1.6 mg / L TDZ, 0.8 mg / L brassinolide solution, 0.5 mg / L methyl jasmonate, 0.8 mg / L IBA, and 0.5 mg / L La(NO3)3 was prepared, and sucrose and agar were completely dissolved in water by heating, and then the mixed solution was added to adjust the pH value to 6.5-6.8, and the volume was fixed and dispensed into tissue culture bottles, and the culture medium was sterilized by high-pressure steam sterilization. After sterilization, it was allowed to stand and solidify to obtain a differentiation medium.

[0104] The clustered bud induction of Example 3 was compared with Comparative Examples 3-4, and the results are shown in Table 4:

[0105] Table 4: Induction of clustered buds in Example 3 and Comparative Example 3-4

[0106] Example 3 Comparative Example 3 Comparative Example 4 Growth coefficient (strain) 5.7 4.6 4.4 Average plant height (cm) 12.8 5.3 8.9 Induction rate (%) 93.67 83.5 78.33 Browning rate (%) 3 2 3

[0107] It can be seen from Table 4 that, compared with Example 3, the proliferation coefficient and the average plant height of Example 3 are significantly reduced, and the induction rate is decreased; compared with Example 3, compared with Example 4, in Example 4, the rape lactone and the culture medium are heated together, which will cause the structure of rape lactone to change at high temperature and weaken the effect.

[0108] Comparative Example 5

[0109] The rejuvenation culture is basically the same as that in Example 3, except that the rejuvenation medium components are different.

[0110] The induced buds are inoculated into a rejuvenation medium, and placed under the conditions of a temperature of 24° C., a light intensity of 6000 Lx, and a light intensity of 16 h / d to induce rejuvenation of the subculture buds for 20-60 days; the rejuvenation medium consists of WPM medium + 1.6 mg / LTDZ + 0.8 mg / LIBA + 0.5 mg / L methyl jasmonate + 3 g / L sucrose + 4.8 g / L agar.

[0111] Comparative Example 6

[0112] The rejuvenation culture is basically the same as that in Example 3, except that the rejuvenation medium components are different.

[0113] The induced buds are inoculated into a rejuvenation medium, and placed under the conditions of a temperature of 24° C., a light intensity of 6000 Lx, and a light intensity of 16 h / d to induce rejuvenation of the subculture buds for 20-60 days; the rejuvenation medium consists of WPM medium + 0.4 mg / LTDZ + 0.6 mg / LIBA + 0.5 mg / L methyl jasmonate + 0.8 mg / L chitin + 3 g / L sucrose + 4.8 g / L agar.

[0114] Comparative Example 7

[0115] The rejuvenation culture is basically the same as that in Example 3, except that the rejuvenation medium components are different.

[0116] The induced buds are inoculated into a rejuvenation medium, and placed under the conditions of a temperature of 24° C., a light intensity of 6000 Lx, and a light intensity of 16 h / d to induce rejuvenation of the subculture buds for 20-60 days; the rejuvenation medium consists of WPM medium + 0.4 mg / LTDZ + 0.6 mg / LIBA + 0.5 mg / L methyl jasmonate + 0.5 mg / L riboflavin sodium phosphate + 3 g / L sucrose + 4.8 g / L agar.

[0117] After 45 days of rejuvenation culture of Example 3 and Comparative Examples 5-7, 5 bottles of rejuvenation tissue culture seedlings were randomly selected from each group to measure their stem diameter, callus conditions at the inoculation site, and proliferation coefficient, as shown in Table 5.

[0118] Table 5 The situation of rejuvenation tissue culture seedlings under different treatments

[0119] Example 3 Comparative Example 5 Comparative Example 6 Comparative Example 7 Culture medium color Light yellow White White Light yellow Stem diameter (mm) 0.93 0.65 0.81 0.73 Callus Yes, very small Yes, larger Yes, smaller Yes, very small Proliferation coefficient 3.1 4.6 3.3 3.6

[0120] As can be seen from Table 5, adding riboflavin sodium phosphate to the rejuvenation medium will make the medium light yellow in color, and adding chitin or riboflavin will reduce the formation of callus at the inoculation site, and the proliferation coefficient will also change accordingly, indicating that the combination of chitin and riboflavin can greatly reduce the formation of callus and the proliferation coefficient.

[0121] The present invention can be implemented in various ways and is not limited to the embodiments described. A person skilled in the art can understand that the present invention can be implemented in other specific ways without changing the technical concept or essential features of the present invention. Therefore, it should be understood that the embodiments described above are exemplary and are not intended to limit the present invention.

Claims

1. A method for promoting the proliferation and rejuvenation of Albizzia julibrissin subgeneration buds, It is characterized in that include: S1, dedifferentiation: 22-generation successive buds were cut into tissue blocks, inoculated into callus induction medium, and induced to dedifferentiate and form callus under preset induction conditions for 14-20 days, wherein the callus induction medium consisted of modified WPM medium, 0.3-0.9 mg / L methyl jasmonate, 0.8-1.6 mg / L TDZ, 0.4-1.2 mg / L NAA, 1.0-2.0 mg / L IBA, 1.5-2.5 g / L sucrose and 4.8-7.5 g / L agar; S2, inducing clustered buds: cutting the callus tissue, inoculating the cut callus tissue into a differentiation medium, and placing it under a preset differentiation condition to induce clustered buds; S3, rejuvenation culture: taking the stems or buds of the clustered buds and inoculating them into a rejuvenation medium, placing them under preset culture conditions and culturing them to obtain a rejuvenation seedling, wherein the rejuvenation medium comprises WPM medium, 0.2-0.6 mg / L LTDZ, 0.4-0.8 mg / L IBA, 0.3-0.8 mg / L methyl jasmonate, 0.5-1 mg / L chitin, 0.1-0.5 mg / L riboflavin sodium phosphate, 3-6 g / L sucrose and 4.8-7.5 g / L agar; S4, subculture: Cut the stem segments of the rejuvenated seedlings and inoculate them into subculture, and culture them under the preset culture conditions.

2. The method for promoting proliferation and rejuvenation of Albizzia julibrissin by claim 1, It is characterized in that The modified WPM medium contains 70-76 mg / L CaCl 2 、470-475mg / LCa(NO 3 ) 2 ·4H 2 O, 170-175mg / LKH 2 PO 4 , 340-360mg / LK 2 SO 4 、360-380mg / LMgSO 4 、180-210mg / L KNO 3 , 0.2-0.3mg / L CuSO 4 ·5H 2 O, 40-50mg / LFeNaEDTA, 2-4mg / LH 3 BO 3 、8-11mg / L MnSO 4 ·H 2 O, 0.1-0.15mg / LNa 2 MoO 4 ·2H 2 O, 10-14mg / LZnSO 4 7H 2 O, 0.5-0.8mg / L niacin, 180-220mg / L inositol, 0.4-0.6mg / L vitamin B 6 and 1-2mg / L vitamin B 1 Made.

3. The method for promoting proliferation and rejuvenation of Albizzia julibrissin by claim 1, It is characterized in that The preset induction conditions include a temperature of 24-28°C, a light intensity of 300-600Lx, and a light duration of 12-14h / d.

4. The method for promoting proliferation and rejuvenation of Albizzia julibrissin by claim 1, It is characterized in that In step S2, the specific steps of cutting the callus tissue and inoculating the cut callus tissue into a differentiation medium are: The callus tissue was cut into 3-5 mm callus blocks, and half of the callus blocks were inserted into a differentiation medium, wherein the differentiation medium was WPM medium, 0.8-2.4 mg / L LTDZ, 0.3-0.8 mg / L methyl jasmonate, 0.4-1.6 mg / L IBA, 0.5-1 mg / L brassinolide, 0.1-0.8 mg / L La(NO 3 ) 3 , 3-6g / L sucrose and 4.8-7.5g / L agar.

5. The method for promoting proliferation and rejuvenation of Albizzia julibrissin subgeneration shoots according to claim 4, It is characterized in that The preparation method of the differentiation medium is as follows: (1) Take WPM medium, TDZ, methyl jasmonate, IBA, La(NO 3 ) 3 A mixed solution is prepared, and sucrose and agar are completely dissolved in water by heating, and then the mixed solution is added to adjust the pH value to 6.5-6.8, and the volume is fixed and divided into tissue culture bottles, and the culture medium is sterilized by high-pressure steam sterilization; (2) preparing a 0.5-1 mg / L brassinolide solution, heating the 0.5-1 mg / L brassinolide solution in a water bath to 75-80° C., and maintaining the temperature for 25-35 min to obtain a sterile 0.5-1 mg / L brassinolide solution; (3) Mix the brassinolide solution with the unsolidified culture medium next to the flame of an alcohol lamp on a clean bench, and let it stand to solidify to obtain a differentiation culture medium.

6. The method for promoting proliferation and rejuvenation of Albizzia julibrissin subgeneration shoots according to claim 1, It is characterized in that The preset differentiation conditions include a temperature of 24-28°C, a light intensity of 3000-4000Lx, and a light intensity of 16-18h / d.

7. The method for promoting proliferation and rejuvenation of Albizzia julibrissin by claim 1, It is characterized in that In step S4, the stem segment is a robust stem with 1-2 nodes retained.

8. The method for promoting proliferation and rejuvenation of Albizzia julibrissin subgeneration shoots according to claim 1, It is characterized in that The subculture medium consists of WPM medium, 0.3-0.8 mg / L methyl jasmonate, 0.6-1.2 mg / L NAA and 0.3-0.6 mg / L TDZ.

9. The method for promoting proliferation and rejuvenation of Albizzia julibrissin subgeneration shoots according to claim 1, It is characterized in that The preset culture conditions include a temperature of 24-28°C, a light intensity of 6000-8000Lx and a light intensity of 16-18h / d.

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